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ORIGINAL ARTICLE
Endophytic Fungal Flora from Roots and Fruits of an IndianNeem Plant Azadirachta indica A. Juss., and Impact of CultureMedia on their Isolation
Vijay C. Verma • Surendra K. Gond •
Anuj Kumar • Ravindra N. Kharwar •
Lori-Ann Boulanger • Gary A. Strobel
Received: 16 February 2009 / Accepted: 24 April 2009 / Published online: 3 February 2011
� Association of Microbiologists of India 2011
Abstract Azadirachta indica A. Juss. (neem), native to
India, is well known worldwide for its insecticidal and
ethanopharmacological properties. Although endophytic
microbes are known from this plant as only leaves and
stems were the subjects of past reports. Now, a variety of
procedures and a number of different media were used to
isolate the maximum number of endophytic fungi from
unripe fruits and roots. A total of 272 isolates of 29 fila-
mentous fungal taxa were isolated at rate of 68.0% from
400 samples of three different individual trees (at loca-
tions-Az1, Az2, Az3). Mycological agar (MCA) medium
yielded the highest number of isolates (95, with a 14.50%
isolation rate) with the greatest species richness. Mycelia
Sterilia (1, 2, 3) accounted for 11.06%, Coelomycetes
7.25%, while Hyphomycetes showed the maximum num-
ber of representative isolates (81.69%). Mycelia-Sterilia
(1, 2, 3), based on their 5.8S ITS 1, ITS2 and partial 18S
and 28S rDNA sequences were identified as Fusarium
solani (99%), Chaetomium globosum (93%) and Chaeto-
mium globosum (93%) respectively. Humicola, Drechslera,
Colletotrichum, and Scytalidium sp. were some of the
peculiar fungal endophytes recovered from this plant.
Keywords Anti-microbial activity � Biodiversity � Fungal
endophytes � Isolation media � Azadirachta indica
Introduction
Azadirachta indica A. Juss (Meliaceae) ‘The Indian Lilac’
having its origin from south East Asia now has worldwide
presence. It is native to India and significantly contributes
to the forest cover of the northern areas. All parts of this
plant show an array of negative effects on insects including
ovipositor deterrent, anti-feedant, and other inhibitory
activities [1, 2]. More than 100 compounds have been
isolated from various parts of the neem tree [3–5] and most
of the active principles (Limnoids) belong to the group of
tetranortriterpinoids especially ‘Azadirachtin’ and its ana-
logs [6]. The people of India have known the useful
properties of neem since time immemorial, and only
recently have other people in more developed countries
realized the value and importance of this tree to human
activity [7]. For instance, various researchers have studied
the medicinal properties of Azadirachta indica including its
anti-pyretic effects [8, 9], anti-malarial effects [10, 11],
anti-tumor effects [12], anti-ulcer effects [13], anti-diabetic
effects [14], anti-fertility effects [15], CNS effects [16],
and cardiovascular effects [17]. Preliminary investigations
of leaves and bark of neem [18–20] cited the fact that
living tissues of neem can successfully harbor endophytic
fungi.
Several reports in the recent years show that the
endophytic fungi from this host produce several bioactive
compounds [21–24]. An endophytic fungus, Phomopsis
sp., isolated from the stems of the neem plant produces
some 10-membered lactones, these lactones have very
promising activity against plant pathogens Ophiostoma
V. C. Verma � S. K. Gond � A. Kumar � R. N. Kharwar (&)
Mycopathology and Microbial Technology Laboratory, Centre
of Advanced Study in Botany Banaras Hindu University,
Varanasi 221005, India
e-mail: rnkharwar@yahoo.com
L.-A. Boulanger
Department of Molecular Biophysics and Biochemistry,
Yale University, 266 Whitney Avenue, New Haven,
CT 06520-8114, USA
G. A. Strobel
Department of Plant Sciences, Montana State University,
Bozeman, MT 59717, USA
123
Indian J Microbiol (Oct–Dec 2011) 51(4):469–476
DOI 10.1007/s12088-011-0121-6
minus and Botrytis cinerea with MIC values 31.25 and
62.50 lg/ml respectively [21]. Again, an endophytic
Geotrichum sp., isolated from the leaves of the neem tree,
has been reported to produce two new chlorinated epi-
meric 1,3-oxazinane derivatives, that have significant
activity against the nematodes Bursaphelenchus xylophi-
lus and Panagrellus redivevus [22]. ‘Javanicin’ an anti-
bacterial nephthaquinone was isolated and characterized
from the endophytic Chloridium sp. obtained from root
tissues of the Azadirachta indica A Juss., this highly
functionalized nephthaquinone exhibits strong antibacte-
rial activity against Pseudomonas spp., representing
pathogens to both humans and plants [23]. Two new
solanapyrone analogues were isolated from the fermen-
tation culture of Nigrospora sp. YB-141, an endophytic
fungus isolated from Azadirachta indica A. Juss. The
structures of the new compounds were elucidated on the
basis of spectroscopic analysis. Most of the compounds
exhibited no or only weak antifungal activities [24]. Thus
with these examples it was established that endophytes
from neem plant have potential bioactive compounds that
need to be characterized.
Thus in anticipation of finding new bioactive com-
pounds we have taken initiative to isolate endophytes from
root and fruits of this vital plant. Endophytic fungi have
been reported from leaves, and stems earlier [20], but in
this report the isolates have been reported from roots and
fruits of neem.
Materials and Methods
Collection Sites and Plant Materials
Varanasi [25.5� N 82.9� E, elevation 279 ft/85 m] is
located into the foothills of Himalayan region of northern
India and has an annual mean temperature of 31�C (max-
imum 38�C, minimum 28�C) with about 110 cm precipi-
tation per annum. Woody perennials dominate the tropical
deciduous forest cover of the region. Three neem plants
(Az1, Az2, Az3) being 10 years old were selected for the
study from this region. The first tree designated-Az1 is
located on the campus of Banaras Hindu University,
Varanasi India. The second tree is Az2 and located in the
forest cover of Chandraprabha Sanctuary range of Varanasi
India, while tree 3-Az3 is located in the Excavation belt of
Sarnath Varanasi India. Roots tissues were recovered by
digging the soil adjacent the main trunk down to 1.5 ft and
root samples, approximately 0.5–1.5 cm diameter and
about 3–5 cm length were collected. The unripe fruits were
collected directly from trees. All samples were then
brought to the laboratory in an icebox, and used to screen
endophytic fungi within 48 h of collection.
Surface Treatment of Plant Samples
To eliminate the epiphytic fungal mycelia, the effective-
ness of various surface decontamination methods was tes-
ted in preliminary experiments. The small segments of
roots (3–5 cm length) and fruits (10 unripe pieces from
each location) were subjected to surface treatments. All
samples were thoroughly washed into running tap water for
about 5–8 min respectively before surface treatments. Two
sterilization regimes were adopted, depending upon vary-
ing the disinfectant and length of surface treatment. In
Regime 1 Sodium Hypochlorite (5.0% w/v), and in Regime
2 H2O2 (35% v/v) were used as surfactants, for 15 min
sterilization length. The samples (50% in five equal parts)
were treated by soaking into 90% ethanol for 1 min, then
placed in sodium hypochlorite (5.0% w/v) for 1, 3, 5, 10,
and 15 min respectively, followed by rinsing in 90% eth-
anol for 10 s. While the remainder of the samples were
treated by soaking into concentrated H2O2 (35% v/v), for 1,
3, 5, 10, and 15 min followed by the same ethanol rinsing
treatment. The samples were then placed on to PDA, and
the plates were placed in an incubator at 25 ± 2�C for at
least 25 days. Tissue (%) from which endophytic mycelia
emerged was recorded each for two treatment conditions at
different lengths of exposure to treatment.
Culture Method and Media
Four culture media used for isolation and identification of
endophytic fungi, were prepared in 1 l each of distilled
water. Malt yeast extract agar (MYA): 10 g malt extract,
2 g yeast extract, 50 mg streptomycin sulphate, 50 mg
chlortetracycline, and 20 g agar. Mycological agar (MCA):
10 g papain-digest of soybean meal, 10 g dextrose, 15 g
agar, 0.4 g cyclohexamide, and 50 mg chloramphenicol.
Difco Potato dextrose agar (PDA): 200 g potato infusion,
dextrose 20 g, and agar 15 g with 100 ppm each of strep-
tomycin sulphate and penicillin. Nutrient agar (NA): 5 g
Peptone, 3 g Beef extract, 5 g NaCl and 15 g Agar. This
medium was specifically chosen for the isolation of acti-
nomycetes and allied genera.
A disc of about 2–3 mm in diameter was cut from the
middle of each root sample, for inoculation. Many discs of
different segments of root samples were taken together
instead from one segment, so as to obtain the greater
diversity of endophytic fungi. This method was also
applied to the fruits samples as well. After surface treat-
ment, 200 discs each from root and fruit samples were
inoculated on to four selected media. All Petri dishes were
sealed with sterile parafilmTM
to protect them from con-
tamination during repeated handling, while examining
endophytes from desiccation. The plates were incubated at
25 ± 2�C and 98% relative humidity (under 12 h
470 Indian J Microbiol (Oct–Dec 2011) 51(4):469–476
123
fluorescent light/12 h dark light), enclosed in translucent
white covered plastic boxes, in BOD cum humidity incu-
bator (L.K Scientific Inc.) for about 25 days.
The emerging endophytic fungi were sub cultured on
PDA, for enumeration and identification. All endophytic
fungal isolates were deposited to the Centre of Advanced
study in Botany, Banaras Hindu University, India [Acces-
sion code: MPCLF/R-1001-1272].
Fungal DNA Isolation and Acquiring ITS-5.8S rDNA
Sequence Information
The fungus was grown on potato dextrose broth for 7 days
and the mycelium was harvested and the nucleic acid (DNA)
was extracted using DNeasy Plant and Fungi Mini Kit
(Qiagen) according to the manufacturer’s directions. The
ITS regions of the fungus were amplified using PCR and the
universal ITS primers ITS1 (50-TCC GTA GGT GAA CCT
GCG G-30) and ITS4 (50-TCC TCC GCT TAT TGA TAT
GC-30). All other procedures were carried out as previously
described by Ezra et al. [25]. The DNA was sequenced at the
W.M. Keck Facility at Yale University. The sequence data of
this fungus are deposited in GenBank and are available on the
NCBI web site (http://www.ncbi.nlm.nih.gov). Sequences
obtained in this study were compared to the GenBank data-
base using the BLAST software on the NCBI web site:
http://www.ncbi.nlm.nih.gov/BLAST/).
Data reduction and Statistics
The isolation frequencies (IR %) were represented by the
ratio of the number of segments/tissues from which the
particular endophytic mycelia emerged and the total seg-
ment/tissues inoculated. One-way analysis of variance
(ANOVA) and Tukey’s multiple range test (TMRT) were
also performed using the SPSS (ver. 10) to determine
which culture media had a significant effect on the isolation
of endophytes.
Results
After consecutive treatments in ethanol/hypochlorite for 1,
3 and 5 min, the relative frequency of tissues from which
endophytic mycelia emerged was calculated. The emer-
gence of endophytic fungi from tissues consistently
increased at 20–30% for 1 min treatment time 37–44% at
3 min, and 40–65% for 5 min from the root and fruits,
respectively (Fig. 1). Thereafter as we increased the length
of treatment time beyond 5 min and there was a gradual
decrease in relative fungal isolation frequency. Interest-
ingly, 35% (v/v) H2O2 (Regime 2) was toxic and seemed
too harsh as some fungal endophytes were killed after 5 min
and during 8–10 min treatment times, the relative frequency
of endophyte isolation dropped significantly up to 50%
(Fig. 1). Overall, the efficacy of 5.0% (w/v) NaOCl treat-
ment for destruction of epiphytes was reaffirmed by the
results. Adding some alcohol can enhance the wetting,
penetrating and killing properties of NaOCl. After opti-
mizing the surface treatment conditions, the method
employing NaOCl at 5% of available chlorine for 5 min
(Regime 1) was then used throughout all remaining exper-
iments. From 400 segments of roots and fruits of
Azadirachta indica, a total of 272 fungal isolates [MCPLF/
R-1001-1272] were recovered representing 29 species of
filamentous fungi (Table 1). Among 272 isolates maximum
165 were obtained from root samples with isolation fre-
quency of 41.25%, while it was only 26.75% in fruits. The
fruit samples exhibit less species richness (20) as compared
to roots (26) (Table 4). From root samples the maximum
number of endophytes was recovered on the PDA medium
(63 isolates, 8 species richness, 28.5% Isolation rate).
However, from fruit samples MCA medium (38 isolates, 6
species richness, and 15% Isolation rates) favors the optimal
recovery of endophytes. Despite the maximum recovery of
endophytes from root tissues on to PDA, the maximum
species richness (11) was obtained on the MCA medium
and similarly with the fruit tissues the maximum species
richness (9) was obtained on the PDA medium despite the
maximum endophytic recovery on MCA medium. Species
such as Chaetomium, Chloridium, Scytalidium, Nigrospora
and Verticillium were exclusively isolated from the root
samples, while Humicola, Drechslera and Colletotrichum
spp. were obtained exclusively from fruits samples irre-
spective of medium used in isolation (Table 1). The maxi-
mum species richness (25 species, 3.27 ± 3.23 species/
media) was obtained from MCA medium. The maximum
numbers of isolates also were recovered on MCA medium
(95). Lowest species richness was obtained with NA med-
ium (26 species, 0.89 ± 1.47 species/media). The number
of species isolated from a particular medium was variable as
Length of sterilization (In min.)
0 2 4 6 8 10 12 14 16 18 20
Tis
sue
(%)
from
whi
ch
endo
phyt
ic m
ycel
ia e
mer
ged
0
10
20
30
40
50
5 % (w/v) NaOCl35 % (v/v) Conc. H2O2
Fig. 1 Influence of surface sterilization on the isolation frequency of
the segments from which endophytic mycelia emerged, depending
upon two sterilents and varying length of sterilization
Indian J Microbiol (Oct–Dec 2011) 51(4):469–476 471
123
reflected by the significant standard deviations (Tables 2,
3). Maximum isolation frequency was obtained on MCA
medium (23.75%) while the least was obtained on the NA
medium (6.50%). Isolation frequencies on PDA (23.25%)
and MCA (23.75%) were almost similar in contrast to MYA
(14.50%) and NA (6.50%) media (Table 3). Acremonium
acutatum, Cladosporium cladosporioides, Curvularia lu-
nata, and Trichoderma sp. were preferentially isolated on
PDA. Alternaria alternata, A. longipes, and Aspergillus
niger, were isolated more frequently on MCA than others.
However, genera like Chloridium virescens and Drechslera
sp. were exclusively isolated on MCA medium. Alternaria
alternata was isolated more frequently on MCA than PDA
and MYA. Alternaria dennisii, Cladosporium cladospo-
rioides, were equally isolated on MYA medium, followed
by Scytalidium and mycelia sterilia (Table 5). Hyphomyc-
ete members also dominated in number over ascomycetes
and zygomycetes (Table 1). Those organisms that were
Table 1 Occurrence of endophytic fungi from roots and fruits of Azadirachta indica on to four different isolation media from three host plants
Root Fruits Endophytic
Fungi Total no. No. of plant amedia of Total no. No. of plant media of of isolates isolation of isolates isolation
Chaetomium crispatum Chaetomium globosum -
Cercinella mucoroides
Colletotrichum Phyllosticta minimaPestalotiopsis
Acremonium acutatumAlternaria alternataAlternaria dennsii Alternaria chlamydosporaAlternaria longipesAspergillus niger Aspergillus fumigatus Aspergillus oryzaeCladosporium cladosporioidesCladosporium acaciicolaCurvularia lunataCurvularia catanulataDrechslera rostrataFusarium oxysporumUlocladium chlamydosporumChloridium virescensHumicola griseaNigrospora oryzaeScytalidiumTrichoderma viridePenicillium cristataVerticillium tenuissimum
FusariumChaetomium globosum
Chaetomium globosum
b
a The symbol represents, for PDA, for MCA, for MYA and for NA
b Fungi that didn’t sporulate on media selected for the study
472 Indian J Microbiol (Oct–Dec 2011) 51(4):469–476
123
listed as Mycelia Sterilia (1, 2, 3) (Table 1) after ITS-5.8 S
rDNA analysis showed that 1 was genetically similar to
Fusarium solani (99%) while 2 was close to Chaetomium
globosum (93%) and 3 was similar to Chaetomium globo-
sum (93%) respectively, with 99–93% sequence similari-
ties. These sequences are deposited in GenBank as
accession EU 80424, EU 780425 and EU 780423, respec-
tively. However, Alternaria along with Aspergillus and
Cladosporium sp., showed their presence on the NA med-
ium as well (Tables 1, 5).
Discussion
The root endophytes are apparently quite common with
their geographic and host distribution, although with the
paucity of morphological characters and intricacy of
inducing sporulation, they were not always easy to identify.
In preliminary experimentation, we optimized the tissue
treatment protocols among the two treatment regimes
(Regimes 1 and 2), because tissue treatment is a crucial
step in endophytic microbe isolation. These regimes differ
in the nature of surface sterilizers (Sterilants) and the
length of treatment. Under Regime 1 the emergence of
endophytic fungi consistently increases from 30 to 65% up
to a sterilization time of 5 min (Fig. 1). Thereafter as the
length of the treatment time increased beyond 5 min there
was a gradual decrease in relative fungal isolation fre-
quency which might be attributed to the enhanced toxicity
of sterilants and sometimes due to the wetting agents
(ethanol in this case), although ethanol has limited pene-
trating and antibiotic activity [26].
Adding some alcohols seems to enhance the wetting,
penetrating and killing properties of NaOCl. Finally, the
efficacy of 5.0% (w/v) NaOCl treatment for destruction of
epiphytes was reaffirmed by the results. Many other reports
also support this conclusion as they find NaOCl at 5% for
about 5 min as the most effective treatment protocol [20],
as in Picea abies the serial washing of root tissues was
done to compare endophytic population, colonizing roots
of the host, in relation to site and soil characteristics [27].
At this point, it can be concluded that the endophytic fungi
which we have recovered, most have tissue specificity
toward the root. This result supports the role of substrate
availability which confined the fungi to specific parts of
host. From root samples, the PDA medium (63 isolates, 8
species richness, 28.5% isolation rate) shows maximum
indices of endophytic recovery in its favor. Similarly, from
fruit samples the MCA medium (38 isolates, 6 species
richness 15.0% isolation rates) favors the optimal recovery
of endophytes. Species such as Chaetomium globosum,
Chloridium, Scytalidium, Nigrospora and Verticillium were
exclusively isolated from the root, while Humicola,
Drechslera and Colletotrichum sp. were obtained exclu-
sively from fruit samples irrespective of media used in
isolation (Tables 1, 4).
Table 2 The effect of culture media on recovery of endophytic
fungal isolates and their respective species richness
Culture
mediaaSegment
cultured
Total isolates
recovered
Species
richness
Species/media
(Mean ± SD)b
MYA 100 58 18 2.00 ± 3.86cd
MCA 100 95 25 3.27 ± 3.23d
PDA 100 93 23 3.20 ± 3.86d
NA 100 26 08 0.89 ± 1.47c
Total 400 272 29 9.37 ± 9.05e
a See materials and method for abbreviationb The values are mean of isolates from three trees at 4 different
culture media. Means followed by different letters (c, d and e) indi-
cates significant difference at P = 0.05, according to one-way
ANOVA and Tukeys multiple range test (TMRT)
Table 3 Host comparisons on recovery of endophytic isolates from
four different culture mediums and their respective isolation
frequency
Culture
mediaaTotal isolates
recovered
Isolation
frequency (%)
Species/media
(Mean ± SD)b
Az1 Az2 Az3
MYA 23 17 18 14.50 2.00 ± 3.86cd
MCA 29 40 26 23.75 3.27 ± 3.23d
PDA 38 25 30 23.25 3.20 ± 3.86d
NA 06 12 08 06.50 0.89 ± 1.47c
Total 96 94 82 68.00 9.37 ± 9.05e
a See materials and method for abbreviationb The values are mean of isolates from three trees at 4 different
culture media. Means followed by different letters indicates signifi-
cant difference at P = 0.05, according to one-way ANOVA and
Tukeys multiple range test
Table 4 A comparison of recovery of endophytic fungi from roots
and fruits of Azadirachta indica
Culture
mediaaIsolates
recovered
Isolation
frequency
Species
richness
Root Fruit Root Fruit Root Fruit
MYA 35 23 17.5 11.5 4 3
MCA 57 38 31.5 15.0 11 6
PDA 63 30 28.5 19.0 8 9
NA 10 16 05.0 08.0 3 2
All media 165 107 41.25 26.75 26 20
a See materials and method for abbreviation
Indian J Microbiol (Oct–Dec 2011) 51(4):469–476 473
123
In case of the MYA medium it was observed that two or
three fast growing fungal species over grew the plate in a
few days and reduced the opportunity for the recovery of
other slow growing fungi. This would be a major factor
resulting in comparatively fewer species (18 species,
2.00 ± 3.86 species/media) from this medium. This may
be attributed to the strong media preference of a few
endophytes than the rest. Thus, media preference is another
factor what can directly influence the endophytic recovery
which was observed in this experiment. The number of
species isolated from a particular medium was variable as
reflected by the significant standard deviations (Table 2).
Some earlier workers also investigated the effects of iso-
lation media on species richness in twigs and leaves of
Chamaecyparis thyoides. It was found that 1% malt extract
and 2% yeast extract with 50 ppm each of Streptomycin
and chlortetracycline gave the highest species richness for
endophytic isolation [28]. One source of variation in the
number of species could be the composition of the med-
ium, as well as suitability of their constituents for fungal
growth. In spite of this fact the maximum isolation fre-
quency was obtained on the MCA medium (23.75%) while
least on to the NA medium (6.50%). Isolation frequencies
of PDA (23.25%) and MCA (23.75%) were almost similar
Table 5 Isolation frequency of endophytic fungi isolated on four different culture media
Isolation frequency (%) Endophytic
Fungi Media preference Root a
Fruit Total (n = 40) (n = 40) (n = 80)
Chaetomium crispatumChaetomium globosumCercinella mucoroidesPhyllosticta minimaPestalotiopsisAcremonium acutatum ColletotrichumAlternaria alternataAlternaria dennisiiAlternaria chamydosporumAlternaria longipesAspergillus nigerAspergillus fumigatusAspergillus oryzae Cladosporium cladosporioides Cladosporium acaciicolaCurvularia lunata Curvularia catinulataDrechslera rostrataFusarium oxysporum Ulocladium chlamydosporumChloridium virescensHumicola griseaNigrospora oryzaeScytalidium Trichoderma viridePenicillium cristataVerticillium tenuissima
a No. of Petri dishes with 5 segments of inoculum’s in each
474 Indian J Microbiol (Oct–Dec 2011) 51(4):469–476
123
in contrast to the MYA (14.50%) and NA (6.50%) media.
Acremonium acutatum, Cladosporium cladosporioides,
Curvularia lunata, Trichoderma sp. were preferentially
isolated on PDA. Alternaria alternata, A. longipes,
Aspergillus niger, were isolated more frequently on MCA
than others. However, genera like Chloridium sp. and
Drechslera sp. were exclusively isolated on MCA medium.
Alternaria alternata was isolated more frequently on MCA
than PDA and MYA. Alternaria dennisii, Cladosporium
cladosporioides, were equally isolated on MYA media,
followed by Scytalidium. Rare or incidental species
(defined in this paper as those species that only represented
by two or three isolates, total 13 species) constitute a high
proportion of overall species richness. These species are
nearly uniform in their recovery from root and fruits
(Table 5). Among fungi recovered in our study Alternaria
sp., Acremonium acutatum, Cladosporium, and Aspergillus
spp. were found to be dominant. Other dominant genera
were Pestalotiopsis, Trichoderma, Curvularia and Peni-
cillium sp. Genera, such as Humicola, Chloridium, Scy-
talidium, and Collitotrichum spp. were obtained for the first
time as endophytes in this plant (Table 1). A considerable
number of strains often remain unidentified in most cases
and this report is no exception to this fact. We too have
recovered Mycelia-Sterilia (MS), which are separated into
three categories based on their morphology. In order to
identify those (MS) we used molecular tools, based on ITS-
5.8 S rDNA sequences followed by BLAST searches. The
mycelia-sterilia (1–3) was identified as Fusarium solani (1)
and Chaetomium Globosum (2, 3) respectively. Leaf iso-
lated mycelia-sterilia 1, was identified as F. solani (soil
fungus), and it shows the vertical traveling nature of this
fungus from root to upper tissues of the host.
Several species we have isolated appear to be rarely
reported from Azadirachta indica including Humicola,
Chloridium, Colletotrichum, and Scytalidium whereas
Choridium and Humicola were reported from twigs of
Terminalia arjuna as well [29]. Many species such as
Alternaria, Colletotrichum, Vertcillium, Aspergillus, and
Penicillium are known to be potential pathogenic fungi to
several hosts and also reported as endophytes to variety of
plants as well. Colletotrichum gloeosporioides is a patho-
genic fungus of Cashew tree, but it was also found as an
endophyte in many cases [25]. Colletotrichum along with
Fusarium were also reported to impair the photosynthetic
activity in Maize and Banana [30] and the presence of
(MS-1) Fusarium solani in this experiment corroborates
this opinion. The exploration of woody perennials for
endophytic microorganisms that might produce microbial
metabolites for use as therapeutic agents needs much
attention. Now with the completion of this work it seems as
if we have a more complete picture of the endophytic
composition of the neem tree when placed in context with
our previous work on neem endophytes [20]. Thus, we are
in a position to carefully screen this array of microorgan-
isms for their bioactive metabolites in order to learn if they
hold as much promise as the host that supports them.
Acknowledgements The authors are thankful to CSIR and UGC
New Delhi for providing financial assistance. Author (Gary Stroble)
expresses his appreciation to the Montana Agricultural Experiment
Station and the US national Science Foundation for their support of
this work. Author (Lori Baulanger) appreciates the H. Hughes grant to
Scott Strobel of Yale University for its support of on this project.
Author (Ravindra Kharwar) is thankful to CSIR for financial assis-
tance (File No.EMR II 05-38/1104) and also to DST New Delhi, for
award of ‘BOYSCAST fellowship’ (SR/BY/L-02/06) 2006–2007).
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